This invention relates to a method for classifying and counting leukocytes by flow cytometry.
In the field of laboratory examination, the classification and counting of leukocytes can give very useful information for diagnosis of disease. Usually, leukocytes in normal peripheral blood come in only five types, lymphocytes, monocytes, eosinophils, basophils and neutrophils. In various hematological disorders, however, immature leukocytes such as myelocytes and myeloblasts appear. Detection of these immature leukocytes is of utmost importance in diagnosing disease.
For this purpose, it has been a customary practice to prepare a blood smear, stain it appropriately, and observe the stained sample microscopically for classification and counting. In recent years, various fully-automated differential leukocyte counters utilizing the principle of a flow cytometer have been produced. These devices can perform highly precise classification and counting of normal leukocytes, but were unable to detect and classify the aforementioned immature leukocytes reliably.
Recent years have seen the provision of reagents and methods which detect the appearance of immature leukocytes highly precisely by the principle of RF/DC measurement (Japanese Laid-Open Patent Publication No. 273413/94).
The method of this publication measures immature leukocytes electrically by use of their nature that under specified conditions, they are less destructible than normal leukocytes. It has also been suggested that their measurement can be made based on information on scattered light. This method, however, aims only at the detection of immature leukocytes. Although it exhibits an excellent performance in the detection of immature leukocytes, the method cannot be used to classify and count normal leukocytes simultaneously with the measurement of immature leukocytes. Alternative methods need to be performed to classify and count normal leukocytes.
Japanese Laid-Open Patent Publication No. 20792/94 discloses a method for blood analysis by causing to leukocytes sufficient damage to allow the passage of a label through them. This method analyzes leukocytes by equally damaging normal leukocytes and immature leukocytes. However, immature leukocytes and normal leukocytes are not necessarily clearly separated according to this method. Furthermore, immature leukocytes treated by this method are permeable to a dye used as the label, and a method which does not cause damage to immature leukocytes is not disclosed in the publication.